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Ultra-VEGF-trap immune fusion protein, its preparation method and application

A fusion protein and protein technology, applied in the fields of peptide/protein components, chemical instruments and methods, hybrid peptides, etc., can solve the problems of reducing half-life, etc., to hinder the transfer pathway, inhibit the formation, and inhibit the development of new blood vessels and lymphatic vessels. generated effect

Active Publication Date: 2013-09-18
JIANGSU JIANDE BIOLOGICAL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The affinity between VEGFR1-Fc and VEGFs is hundreds of times higher than that of VEGFR2-Fc (Kuo, C. et al. PNAS, 2001 98, 4605-4610), but VEGFR1-Fc contains more basic amino acids, and its isoelectric point is greater than 9.5 , it is easier to combine with acidic matrix in vivo, reducing its half-life in blood (2000, Cancer Res.60, 6253-6258.)

Method used

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  • Ultra-VEGF-trap immune fusion protein, its preparation method and application
  • Ultra-VEGF-trap immune fusion protein, its preparation method and application
  • Ultra-VEGF-trap immune fusion protein, its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1 Constructing the expression vector of the immune fusion protein Ultra-VEGF-trap

[0025] 1. Synthesis of AmFc1 and BmFc2 genes

[0026] Entrusted Shanghai Jierui Bioengineering Co., Ltd. to synthesize the AmFc1 coding gene sequence, its nucleotide sequence is shown in SEQ ID.2; the amino acid sequence of AmFc1 protein is shown in SEQ ID.3, including signal peptide sequence, VEGFR1 extracellular Two regions, the third extracellular functional region of VEGFR2, and the constant region Fc of human IgG1, in which Fc has undergone two amino acid mutations, the E at position 357 is mutated to K, and the D at position 399 is mutated to K. Named AmFc1.

[0027] Also commissioned Shanghai Jierui Bioengineering Co., Ltd. to synthesize the BmFc2 coding gene sequence, the nucleotide sequence is shown in SEQ ID.4; the amino acid sequence of BmFc2 protein is shown in SEQ ID.5, including the signal peptide sequence, VEGFR3 extracellular 1 region, the 2nd functional region an...

Embodiment 2

[0031] Example 2 Expression and purification of immune fusion protein Ultra-VEGF-trap

[0032] 293F (purchased from Invitrogen, Cat No. 11625-019) cells were suspended and cultured in serum-free CD293 medium (purchased from Invitrogen, Cat No. 11913-019), centrifuged before transfection to replace fresh medium, and the cell concentration was adjusted It is 1×106 cells / ml. Taking 100ml of cells as an example, add 150μg of DNA (pT1h-ONC) and 300μg of PEI (Sigma, Cat. No: 408727) into 10ml of 293 culture medium, mix well, and let stand for 5min. After standing at room temperature for 20 minutes, the PEI / DNA suspension was added dropwise to the shake flask, mixed gently, placed in 5% CO2, 37° C. shaker culture (115 rpm) for 5 days, and the culture supernatant was collected.

[0033] Five days after transfection, the supernatant was collected for purification. Equilibrate HiTrap MabSelectSuRe 1ml column (GE Healthcare Life Sciences product, Cat.No: 11-0034-93) 10 bed volumes with...

Embodiment 3

[0035] Example 3 Functional analysis of the immune fusion protein Ultra-VEGF-trap

[0036] VEGF165 (Beijing Hongyue Innovation Technology Co., Ltd., cat: 100-20), VEGF-B (Beijing Hongyue Innovation Technology Co., Ltd., cat: 100-20B), VEGF-C (Beijing Hongyue Innovation Technology Co., Ltd., cat: 100-20C), VEGF-D (Beijing Hongyue Innovation Technology Co., Ltd., cat: 100-20D) were diluted with 0.05mmol / L sodium carbonate, sodium bicarbonate buffer (pH 9.6) to 2μg / mL, 100μL / The wells were coated overnight at 4°C; after blocking with 1% PBS-BSA at room temperature for 1 hour, different concentrations of fusion proteins were added and incubated at room temperature for 2 hours. After washing with PBST (0.05% Tween-20) for 3 times, add 1:30000 dilution of alkaline phosphatase-labeled anti-human IgG (Zhongshan Jinqiao, cat: ZB2304), 100 μL / well, incubate at room temperature for 45 min; wash 3 times with PBST Afterwards, 100 μL / well of TMD substrate color developing solution (Kangwe...

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Abstract

The invention provides an Ultra-VEGF-trap immune fusion protein, which can be the protein in 1) or 2): 1) a protein composed of the amino acid sequence shown in SEQID.1; and 2) an anti-tumor related protein that is derived from 1) and is obtained by subjecting the amino acid sequence shown in SEQID.1 to substitution and / or deletion and / or adding by one or several amino acids. The invention also provides a construction method and application of the Ultra-VEGF-trap immune fusion protein. The immune fusion protein provided in the invention totally blocks the transduction pathway of VEGFs / VEGFRs, and has enormous value for treatment of multiple malignant tumors.

Description

technical field [0001] The invention relates to an Ultra-VEGF-trap immune fusion protein, a preparation method and application thereof, and belongs to the field of biotechnology. Background technique [0002] Unlimited invasive growth of tumors and their metastasis are dependent on angiogenesis. If there is no new blood vessels to supply nutrients, the tumor volume will not exceed 2mm 3 -3mm 3 . Tumor angiogenesis can be divided into prevascular phase and vascular phase. In the prevascular stage, tumor cells mainly rely on diffusion to support and excrete metabolites. Although tumor cells proliferate rapidly, proliferation and apoptosis are in a dynamic balance, and the tumor size generally does not exceed 1-2mm 3 , can remain months or years without transfer. Once the tumor transforms into the angiogenesis phase, the tumor angiogenesis factors are overproduced and out of balance with the inhibitory factors, blood and nutrients are supplied through angiogenesis, apopto...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K38/16A61P35/00
Inventor 胡品良
Owner JIANGSU JIANDE BIOLOGICAL PHARMA
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